A reversible, switchable pH-driven quaternary ammonium pillar[5]arene nanogate for mesoporous silica nanoparticles

Article abstract of DOI:10.1039/c9tb00946a

Here we describe the assembly and pH-driven operation of two nanocarriers based on non-functionalized (MCM-41) and carboxylate-functionalized (MCM-41-COOH) containers loaded with the anticancer drug doxorubicin (DOX) and capped by quaternary ammonium pill

Full text of DOI:10.1039/c9tb00946a

Journal of
Materials Chemistry B
View Article Online
View Journal
PAPER
A reversible, switchable pH-driven quaternary
ammonium pillar[5]arene nanogate for
mesoporous silica nanoparticles†
Cite this:DOI: 10.1039/c9tb00946a
a
a
a
Evelyn C. S. Santos,
Fernanda L. Jorge,
Pamella S. Cordeiro,
ˆ
Thiago C. dos Santos,
Tamires S. Fernandes,
Vinicius G. C. Madriaga,
Nathalia Meireles Da Costa,
c
d
a
Vanessa Nascimento,
d
b
c
Noemi R. Checca,
c
a
Luıs Felipe Ribeiro Pinto
and Celia M. Ronconi
*
´
´
Here we describe the assembly and pH-driven operation of two nanocarriers based on non-functionalized
(MCM-41) and carboxylate-functionalized (MCM-41-COOH) containers loaded with the anticancer drug
doxorubicin (DOX) and capped by quaternary ammonium pillar[5]arene (P[5]A) nanogates. MCM-41 and
MCM-41-COOH containers were synthesized and transmission and scanning electron microscopies
showed nanoparticles with spherical morphology and dimensions of 85 ꢀ 13 nm. The nanochannels of
MCM-41 loaded with DOX were gated through the electrostatic interactions between P[5]A and the
silanolate groups formed at the silica–water interface, yielding the MCM-41-DOX-P[5]A nanocarrier. The
second nanocarrier was gated through the electrostatic interactions between the carboxylate groups
mounted on the surface of MCM-41 and P[5]A, resulting in the MCM-41-COO-DOX-P[5]A nanocarrier.
The DOX release profiles from both nanocarriers were investigated by UV-vis spectroscopy at different pH
values (2.0, 5.5 and 7.4) and also in the presence of ions, such as citrate3ꢁ (19 mmol Lꢁ1) and Zn2+ (1.2 and
50 mmol Lꢁ1) at 37 1C. MCM-41-COO-DOX-P[5]A can be turned on and off eight times through the
formation and breaking of electrostatic interactions. In vitro studies show that MCM-41-COO-DOX-P[5]A
can penetrate and release DOX in the nucleus of human breast adenocarcinoma MCF-7 cancer cells
leading to a pronounced cytotoxic effect. Therefore, the fabricated nanocarrier based on a water-soluble
cationic pillar[5]arene nanogate, which is reversibly opened and closed by electrostatic interactions, can be
considered as a promising drug transport and delivery technique for future cancer therapy.
Received 10th May 2019,
Accepted 6th December 2019
DOI: 10.1039/c9tb00946a
rsc.li/materials-b
components, which reversibly open and close the nanochannel
entryways of the MSNPs to allow content release by an external
Introduction
The use of gated nanocarriers for the smart controlled release of stimulus,4–9 such as light/heat irradiation,10–12 a pH change,4,13,14
chemotherapy drugs has attracted considerable attention as a alternating magnetic field application,15 and enzymatic16 and
potential alternative system for cancer treatment.1–3 These systems redox reactions.17,18
can be built by attaching supramolecular binding stations on
Various macrocycles can be used as moving components or
mesoporous silica nanoparticles (MSNPs) to interact with moving nanogates to seal the nanochannels and prevent content leak-
age, e.g., cyclodextrins,18 crown ethers,19 cucurbiturils2 and
calixarenes.20 Pillar[n]arenes are emerging as a novel class of
a
´
ˆ
Departamento de Quımica Inorganica, Universidade Federal Fluminense,
synthetic macrocycle paracyclophane derivatives that consist of
1,4-disubstituted hydroquinone units linked by methylene bridges
in their para-positions. They have an electron-rich cavity and high
symmetry and can be chemically modified at both the upper and
lower rims in contrast to other macrocycles.21–26 The first water-
soluble pillar[5]arene was synthesized in 2010 by introducing
carboxylate groups around the rims of a macrocycle.27 Water-
soluble pillararenes have considerable applications in the field
of mechanochemistry.28–30 For example, a carboxylic acid-
substituted pillar[5]arene (CP[5]) has been used as a moving
˜
˜
Campus do Valonguinho, Outeiro Sao Joao Batista s/n, Centro,
´
24020-150, Niteroi, RJ, Brazil. E-mail: cmronconi@id.uff.br
b
c
´
Centro Brasileiro de Pesquisas Fısicas (CBPF), Dr Xavier Sigaud 150, Urca,
22290-180, Rio de Janeiro, RJ, Brazil
ˆ
Molecular Carcinogenesis Program, Instituto Nacional de Cancer (INCA),
´
Andre Cavalcanti 37, Centro, 20231-050, Rio de Janeiro, RJ, Brazil
d
´
ˆ
Departamento de Quımica Organica, Universidade Federal Fluminense,
˜
˜
Campus do Valonguinho, Outeiro Sao Joao Batista s/n, Centro, 24020-150,
´
Niteroi, RJ, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9tb00946a
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
component in nanovalves.4,13,14,31–38 However, the water-soluble The capacity of MCM-41-COO-DOX-P[5]A to deliver doxorubicin
quaternary ammonium pillar[5]arene (P[5]A) has not been exten- inside human breast adenocarcinoma MCF-7 cancer cells was also
sively explored, and to the best of our knowledge, it has not been evaluated.
reported in the literature as a nanogate. Cationic P[5]A has the
advantage of interacting electrostatically with the negatively
charged surface of MSNPs through the conjugate base silanolate
that forms at the silica–water interface; thus, functionalization may
not be required, eliminating one step in the assembly of the
Results and discussion
Synthesis and characterization of P[5]A, MCM-41, MCM-41-CN
and MCM-41-COOH
nanodevice.
The cationic pillar[5]arene was synthesized using the general
In this work, we explored the use of the water-soluble quaternary procedure introduced by Ogoshi.22,39 Typically, hydroquinone
ammonium pillar[5]arene P[5]A, as a nanogate in the construction bis(2-hydroxyethyl)ether was converted to the brominated
of two nanocarriers. P[5]A was employed as a stopper to seal the hydroquinone 1 by the Appel reaction. Then, the cyclization
nanochannels of MSNPs through simple electrostatic interactions of compound 1 with paraformaldehyde was carried out in the
between the positively charged ammonium groups located on the presence of the Lewis acid BF3ꢂOEt2 and the template dichloro-
upper and lower rims of P[5]A and the negatively charged surface of methane to yield brominated pillar[5]arene 2. Compound 2 was
the MSNPs. We assembled the first nanocarrier by mounting the converted to the quaternary ammonium pillar[5]arene named
P[5]A nanogate directly on non-functionalized MCM-41 (MCM-41- P[5]A by reacting with trimethylamine in ethanol (Scheme S1,
DOX-P[5]A) and the second one by mounting P[5]A on carboxylate ESI†).39 The product P[5]A was characterized by 1H and 13C
group-functionalized MCM-41 (MCM-41-COO-DOX-P[5]A). The NMR spectroscopy (Fig. S1 and S2, ESI†).
nanocarriers were loaded with doxorubicin (DOX), and the release
MCM-41 was synthesized by a base-catalyzed sol–gel method
of the drug was investigated (Scheme 1). P[5]A was found to strongly using a cationic surfactant (hexadecyltrimethylammonium bromide,
interact with the silanolate groups of non-functionalized MCM-41 CTAB) template following the procedures described by Zink et al.40–42
and not fully disconnect on command (acid pH). In contrast, To functionalize MCM-41 with carboxyl groups we used a
P[5]A coupled to carboxylate group-functionalized MCM-41 methodology employed by our group to modify silica nano-
(MCM-41-COO-DOX-P[5]A) not only obeys commands but also particles with various functional groups.17,18,43–50 MCM-41 con-
can be reversibly switched eight times in response to pH changes. taining the CTAB template (MCM-41-CTAB) in the pores was
Scheme 1 Schematic representation of the preparation of DOX-loaded MCM-41-COOꢁ and its interaction with the positively charged P[5]A nanogate.
The same procedure was carried out for non-functionalized MCM-41. MCM-41-COO-DOX-P[5]A and MCM-41-DOX-P[5]A can be operated by
chemical stimulus (pH changes) to regulate the release of doxorubicin (DOX).
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Journal of Materials Chemistry B
Paper
modified with 3-cyanopropyltriethoxysilane (CyPTES) to form scattering (DLS) are 170.3 and 211.9 nm, respectively, indicating that
MCM-41-CTAB-CN, followed by the acid-catalyzed hydrolysis of both materials are well dispersed in aqueous medium (Fig. S10,
the 3-cyanopropyl groups to produce 3-carboxypropyl groups on ESI†).
the surface of the MCM-41. CTAB template extraction from the
pores was carried out using ethanolic ammonium chloride to MCM-41-COOH are ꢁ14.8 and ꢁ30.2 mV at pH = 7.4, respec-
form the compound named MCM-41-COOH. tively (Fig. S11, ESI†). The z-potential values of both MCM-41
The zeta potential (z-potential) values of MCM-41 and
The FTIR spectra of MCM-41-CTAB, MCM-41, MCM-41-CN, and MCM-41-COOH become more negative with increasing
and MCM-41-COOH show bands at 1060 and 803 cmꢁ1, which pH due to the deprotonation of the silanol and carboxypropyl
are ascribed to the asymmetric and symmetric stretching of groups, respectively. MCM-41-COOH shows more negative
Si–O–Si bonds, respectively (Fig. S3, ESI†). The band at 947 cmꢁ1 z-potential values than MCM-41 because the carboxypropyl
is due to the Si–OH stretching mode.51 The bands at 2926 and groups are more acidic than the silanol groups; therefore, more
2852 cmꢁ1 and the band in the region of 1400–1500 cmꢁ1 of the deprotonated species form on its surface.
spectrum of MCM-41-CTAB are assigned to the C–H stretching
To confirm the higher acidity of MCM-41-COOH compared
and C–H bending vibration modes of methyl and methylene to that of MCM-41, potentiometric titrations were carried out
groups of CTAB.52 The absence of the bands related to CTAB in (Fig. S12, ESI†). The Gran plots (Fig. S13, ESI†) show that the pKa
both the MCM-41-CN and MCM-41-COOH materials indicates and the concentration of the acid sites are 7.2 and 154 mmol gꢁ1 for
that the template was successfully removed. The Raman spectrum of MCM-41 and 6.1 and 582 mmol gꢁ1 for MCM-41-COOH (Table S2,
MCM-41-CN shows a weak band at 2249 cmꢁ1 due to the CRN ESI†).59,60 Therefore, functionalization with carboxypropyl groups
stretching vibration mode (Fig. S4, ESI†).53,54 The band at 1717 cmꢁ1 improved the acidity of MCM-41, as expected.
in the FTIR spectrum for MCM-41-COOH is due to the CQO
FTIR spectroscopy of DOX-unloaded MCM-41-P[5]A and
MCM-41-COO-P[5]A
stretching vibration mode, confirming the oxidation of 3-cyano-
propyl groups into 3-carboxyproyl groups.55 The oxidation of
the cyano groups was also confirmed by elemental analysis, Before loading the nanochannels of the MSNPs with DOX, the
which showed carbon and nitrogen contents of 8.3 and electrostatic interactions between the cationic pillar[5]arene
1.9 mmol gꢁ1, respectively, for MCM-41-CN. After the hydrolysis P[5]A and MCM-41 and MCM-41-COOH were investigated by
of the cyano groups, the carbon content reduced to 5.8 mmol gꢁ1 FTIR spectroscopy (Fig. S14, ESI†). The P[5]A salt shows absorp-
and 0.3 mmol gꢁ1 of nitrogen was detected in the MCM-41- tion bands at 1612 cmꢁ1 and 1486 cmꢁ1 due to the phenyl
COOH sample. Most likely, the reduction in the carbon content skeleton stretching vibration CQC, 1204 and 1063 cmꢁ1 related
is related to the possible leaching of organosilane chains from to the asymmetric and symmetric stretching vibration of aryl
the surface of MCM-41-COOH during the hydrolysis step.
alkyl ether C–O–C.61–63 The absorption band at 1717 cmꢁ1
,
The nitrogen adsorption–desorption isotherms of MCM-41 assigned to the CQO stretching vibration of the carboxylic acid
are type IV(b), indicating the presence of cylindrical mesopores group of MCM-41-COOH, disappears in MCM-41-COO-P[5]A
with a diameter smaller than 4 nm.56,57 This material exhibits a due to the formation of carboxylate groups that electrostatically
surface area of 1034 m2 gꢁ1, a pore volume of 1.14 cm3 gꢁ1 and interact with P[5]A. We should expect the absorption bands at
a narrow pore distribution of 2.72 nm based on the Barrett– 1650 and 1400 cmꢁ1 to be related to the asymmetric and symmetric
Joyner–Halenda (BJH) method (Fig. S5, S6 and Table S1, ESI†). vibration modes, respectively, of (COO)ꢁ in MCM-41-COO-P[5]A;
PXRD analysis of MCM-41 shows three reflection angles at 2y = however, these absorption bands may overlap with the quaternary
2.15, 3.71 and 4.30, indexed as (100), (110) and (200) Bragg ammonium P[5]A absorption bands.
peaks, respectively (Fig. S7, ESI†). The (100) reflection is asso-
Colloidal studies on the interaction between cationic P[5]A and
MCM-41 and MCM-41-COOH
ciated with the hexagonal structure of MCM-41, and the other
reflections are due to the crystallinity.58 Scanning electron
microscopy (SEM) of MCM-41 shows spherical particles with a The interactions between P[5]A and MCM-41 and MCM-41-
size distribution centered at 85 nm ꢀ 13 nm (Fig. S8, ESI†). COOH were also investigated by measuring the zeta potential
Transmission electron microscopy (TEM) shows a well-ordered (z-potential) changes of their dispersions in phosphate buffered
hexagonal mesoporous structure of MCM-41 (Fig. S8, ESI†).
saline (PBS) solutions containing 0.5 mg mLꢁ1 of MCM-41 or
Upon functionalization of MCM-41 with carboxyl groups MCM-41-COOH as a function of the P[5]A concentration
(MCM-41-COOH), the nitrogen adsorption–desorption isotherms (Fig. 1).
remain of type IV(b), thus the mesoporous structure was not
As previously discussed (Fig. S11, ESI†), at pH 7.4, MCM-41
damaged (Fig. S5, ESI†); however, the surface area, pore volume and MCM-41-COOH are negatively charged because silanolate
and pore diameter decreased to 755 m2 gꢁ1, 0.97 and 2.54 nm, and carboxylate groups form at the interface between the
respectively (Table S1, ESI†). The PXRD pattern of MCM-41- solids and water; both can electrostatically interact with P[5]A. Prior
COOH shows a decrease in all peak intensities, suggesting a to the addition of P[5]A, the z-potential values of MCM-41 and
crystallinity reduction (Fig. S7, ESI†). The TEM images clearly show MCM-41-COOH were ꢁ14.8 and ꢁ30.2 mV, respectively, at pH 7.4
that the well-ordered hexagonal mesoporous structure remains and room temperature (Fig. 1 and S11, ESI†). The isoelectric
after the acid hydrolysis process (Fig. S9, ESI†). The diameters points (IEPs) of the MCM-41 and MCM-41-COOH dispersions
of MCM-41 and MCM-41-COOH measured by dynamic light were reached at a concentration of 86.8 mg Lꢁ1 of P[5]A (Fig. 1).
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
between P[5]A and the surfaces (Fig. 2). The z-potential values
gradually decrease upon the acidification of the dispersions due
to the protonation of the silanolate or carboxylate groups (Fig. 2).
The IEP of the MCM-41-COO-P[5]A dispersion is at pH = 2.6. This
result demonstrates that at an acidic pH, the protonation of the
carboxylate groups in MCM-41-COO-P[5]A occurs because the
surface charge shifts to zero, releasing the macrocycle P[5]A into
the solution.
For MCM-41-P[5]A, the z-potential values are positive over
the entire pH range (2.0–12.0), indicating that P[5]A remains on
MCM-41 even under very acidic conditions (pH = 2.1). The inner
diameter of the paracyclophane moiety of the optimized structure
of P[5]A is 0.84 nm, and the distance between the two opposite
quaternary ammonium groups located on the same side of the
rim is 1.64 nm (Fig. S15, ESI†). Therefore, P[5]A can also interact
with the silanolate groups located inside the pores of the
unloaded MCM-41. Upon the addition of acid, the silanolate
groups on the external surface of MCM-41 are easily protonated,
and the P[5]A stoppers are released. However, those located inside
the empty pores remain.
Fig. 1 z-Potential measurements as a function of P[5]A concentration for
MCM-41 and MCM-41-COOH at pH = 7.4 and RT in PBS buffer.
Higher concentrations of P[5]A shifted the z-potential to positive
values, and then it became almost constant at concentrations
higher than 200 mg Lꢁ1 for both materials. The positive values of
the z-potential are related to the positive P[5]A-silica–water inter-
face (named MCM-41-P[5]A or MCM-41-COO-P[5]A), in which the
non-interacting rims of P[5]A provide the surface charge in
colloidal suspensions. Additionally, for concentrations above
200 mg mLꢁ1 of P[5]A, the z-potential values did not significantly
change because the negative surfaces of MCM-41 and MCM-41-
COOH were saturated with P[5]A, and the excess of P[5]A did not
interact with the MSNP surfaces.
The effects of pH (2.0–12.0) on the interactions between
P[5]A and MCM-41 or MCM-41-COOH were also investigated by
z-potential measurements (Fig. 2). The z-potential values of
MCM-41 and MCM-41-COOH suspensions in PBS, both capped
with P[5]A (MCM-41-P[5]A or MCM-41-COO-P[5]A), are +21.4 and
+11.1 mV, respectively, at pH 7.4 as a result of the interactions
Characterization of DOX-unloaded and loaded nanocarriers by
N2 adsorption–desorption isotherms
The amounts of DOX loaded in MCM-41-DOX-P[5]A and MCM-
41-COO-DOX-P[5]A were obtained from a DOX standard curve
monitoring the DOX absorption band at 483 nm (Fig. S16,
ESI†).17,26,64 The calculated amounts of loaded DOX were
27.2 mg gꢁ1 (MCM-41-DOX-P[5]A) and 28.6 mg gꢁ1 (MCM-41-
COO-DOX-P[5]A) corresponding to a DOX-loading efficiency of
52.4% and 57.6%, respectively. For the detailed calculations,
see the Experimental section.
To investigate the effect of the P[5]A stopper and the drug on the
mesopore structure of the MSNPs, the N2 adsorption–desorption
isotherm curves of the DOX-unloaded (MCM-41-P[5]A and MCM-
41-COO-P[5]A) and DOX-loaded nanocarriers (MCM-41-DOX-P[5]A
and MCM-41-COO-DOX-P[5]A) were measured. The isotherms of
MCM-41-P[5]A and MCM-41-COO-P[5]A showed type III profiles,
which are characteristic of nonporous materials (Fig. S5 and
Table S1, ESI†). Condensation and evaporation phenomena in
the region of 0.2 bar o p/p0 o 0.4 bar are absent in these
isotherms indicating complete mesopore obstruction due to the
presence of the P[5]A stopper. Similar behaviors were observed
for the isotherms of DOX-loaded MCM-41-DOX-P[5]A and MCM-
41-COO-DOX-P[5]A nanocarriers (Fig. S5, ESI†). The BJH pore
size distributions confirmed the absence of mesopores in the
DOX-unloaded and loaded nanocarriers (Fig. S6, ESI†).
pH-Trigged DOX release from MCM-41-DOX-P[5]A and
MCM-41-COO-DOX-P[5]A
The efficiency of P[5]A in blocking the nanochannel entryways
on MCM-41 and MCM-41-COOH and avoiding drug release at
physiological conditions (37 1C and pH 7.4) was evaluated by
monitoring the absorption band of DOX at 483 nm over time in
the UV-vis spectrum. The amounts of DOX released were
calculated from its standard curve (Fig. S16, ESI†). The DOX
leakage in the capped MCM-41-COO-DOX-P[5]A nanocarrier is
Fig. 2 z-Potential measurements as function of pH for MCM-41-P[5]A
and MCM-41-COO-P[5]A.
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Journal of Materials Chemistry B
Paper
lower (4%) than that observed for MCM-41-DOX-P[5]A (11%)
within 30 h. These are minimal and acceptable leakages, as
reported in the literature for other systems.16,19,65 Therefore,
these findings demonstrate that the water-soluble quaternary
ammonium pillar[5]arene, P[5]A, can block the nanochannel
entryways on MCM-41 and MCM-41-COOH, leading to low DOX
leakage under physiological conditions particularly for the
carboxyl functionalized material.
To release DOX from MCM-41-DOX-P[5]A and MCM-41-COO-
DOX-P[5]A, the pH was adjusted from 7.4 (physiological pH) to
5.5 (lysosomal pH) and 2.0 by adding calculated volumes of
dilute HCl. These studies were carried out at 37 1C (body
temperature) and the absorption band of DOX at 483 nm in
the UV-vis spectrum was monitored over time (Fig. S17, ESI†).
As shown in Fig. 2, under acidic conditions the silanolate
(MCM-41) or carboxylate groups (MCM-41-COOꢁ) are protonated,
and the electrostatic interactions with cationic P[5]A are weakened,
opening the nanogate.
Fig. 3 shows the DOX release profiles as percentages versus
time. At physiological pH (7.4), 37 1C (Fig. 3) and before HCl
addition, MCM-41-DOX-P[5]A and MCM-41-COO-DOX-P[5]A are
in the ‘‘gate-closed state’’, and the DOX leakage monitored
within 2 h was only 4.1 and 1.7%, respectively. After 2 h, HCl
was added to the cuvette to reach pH = 5.5 or 2.0. At this point,
the nanocarriers immediately switched to the ‘‘gate-open state’’
and a burst release of DOX was detected (Fig. 3 and Fig. S17,
ESI†). The kinetics is faster in the first stages of DOX release for
both nanocarriers at both pH values. At acid pH values, the
silanolate or carboxylate conjugate bases of MCM-41 and MCM-
41-COOꢁ are immediately protonated and the equilibrium
shifts towards the nonionized acid form (silanol and carboxyl
groups). The outcome is that the P[5]A molecules located on the
surface of MCM-41 promptly dissociate and DOX is released.
However, at pH = 2.0 the equilibrium is reached more quickly
for MCM-41-DOX-P[5]A and the percentage of DOX released
from this nanocarrier (59%) is smaller than that released by
Fig. 3 Acid-triggered release profiles of DOX in PBS from (a) MCM-41-
DOX-P[5]A and (b) MCM-41-COO-DOX-P[5]A at 37 1C as a function of
time.
MCM-41-COO-DOX-P[5]A (91%) in the same period of time (30 h) electrostatically interacts with the negatively charged surface of
(Fig. 3). We can explain the behavior of MCM-41-DOX-P[5]A MCM-41-COOꢁ (Fig. 1). With addition of acid (pH = 5.5 or 2.0) the
based on the results in Fig. 2, which shows that P[5]A is not carboxylate groups are protonated and the attractive coulombic
totally removed from the DOX-unloaded MCM-41-P[5]A nano- forces are broken, releasing the stopper and drug simultaneously.
carrier, most likely because part of the P[5]A is interacting with
silanolate groups inside the pores of MCM-41, making them less COOH could act as chemical inputs, regenerating the electrostatic
accessible for protonation.
interaction between P[5]A and MCM-41-COOꢁ and ceasing the
Bases that can deprotonate the carboxyl groups of MCM-41-
Therefore, due to the lower release of DOX from MCM-41- release of DOX from the nanochannels of the MSNPs. Therefore,
DOX-P[5]A compared to MCM-41-COO-DOX-P[5]A, the next the chemical switching of the nanogate was investigated by succes-
studies were carried out using the latter system.
sive additions of HCl to a suspension of MCM-41-COO-DOX-P[5]A
in PBS (pH = 7.4) to reach pH 5.5 or 2.0 and then addition of NaOH
to return to pH 7.4 (Fig. 4). Upon addition of calculated volumes of
3.0 mol Lꢁ1 HCl to the dispersion in PBS (the pH changes from 7.4
Acid- and base-driven chemical switching of MCM-41-COO-
DOX-P[5]A
To demonstrate that P[5]A behaves as a switchable nanogate in to 5.5 or 2.0, first point of the curve in Fig. 4) the nanogate is turned
MCM-41-COO-DOX-P[5]A which can be turned on and off to regulate on, and DOX is immediately released (Fig. 4 and Fig. S18, ESI†).
the passage of DOX through the nanochannels of MCM-41 functio- Subsequently, the nanogate is turned off by adjusting the pH to 7.4
nalized with carboxyl groups, we carried out an experiment based on through the addition of a NaOH solution (3.0 mol Lꢁ1). In this
the reversibility of the acid–base equilibrium between the carboxyl state, the nanogate is closed, and the DOX release ceases; e.g.,
group (MCM-41-COOH) and the carboxylate anion (MCM-41-COOꢁ). the percentage of DOX released remains almost constant.
We have already demonstrated that the cationic stopper P[5]A The switching process was repeated eight times, proving that
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 5 The release profiles of DOX from MCM-41-COO-DOX-P[5]A in the
presence of citrate3ꢁ (19 mmol Lꢁ1) and Zn2+ (1.2 and 50 mmol Lꢁ1) in PBS
buffer solutions at 37 1C.
Fig. 4 DOX release profiles as a function of time from MCM-41-COO-
DOX-P[5]A after consecutive additions of acid and base to a suspension of
the nanocarrier in PBS at 37 1C.
The concentrations of Zn2+ are in the range of nano to picomolar
depending on the type of tissue and methodology of analysis.72–74
Because the surface of the mesoporous silica contains carboxylate
groups, Zn2+ could compete with P[5]A by these negative groups
and turn-on the nanogate, leading to premature release. Fig. 5 and
Fig. S19 (see the ESI†) show the release profiles of DOX from MCM-
41-COO-DOX-P[5]A in water upon addition of Zn2+ at two concen-
MCM-41-COO-DOX-P[5]A operates almost reversibly. In theory,
the reversibility of the switching mechanism allows this nano-
carrier to be reloaded with DOX in the gate-open state, re-locked
in the gate-closed state and reused at least eight times.
Investigation of competitive binding using citrate3ꢁ and Zn2+
The following experiments were carried out to investigate the trations (1.2 and 50 mmol Lꢁ1). The release of DOX to the solution
effect of some ionic species which are present in the blood on using 1.2 mmol Lꢁ1 and 50 mmol Lꢁ1 was 7 and 10%, respectively.
the nanogate operation. There is a myriad of ionic species that Therefore, Zn2+ cations are not able to efficiently open the nano-
could affect the nanogate operation leading to premature gate. Most likely, there is an electrostatic repulsion between Zn2+
release of the drug before reaching the target, and it would cations and the positive surface of the nanogate in MCM-41-COO-
be impossible to investigate all of them. Therefore, we choose DOX-P[5]A (see the z-potential values in Fig. 1), preventing Zn2+
to investigate the effect of some ionic species present at binding to carboxylate groups anchored on the surface of MCM-41.
considerable concentrations in the blood, such as citrate3ꢁ
and Zn2+
Therefore, neither citrate3ꢁ nor Zn2+ at the investigated
concentrations were able to efficiently open the nanogate,
.
High levels of citrate ions are naturally present in most which is an interesting result for practical applications because
healthy cells due to their vital importance in the Krebs cycle or the undesirable release of DOX in the presence of these ions in
citric acid cycle.66–68 In the blood, citrate acid exists predo- the blood would decrease the ability of the nanocarrier to treat
minantly as a trivalent anion (citrate3ꢁ) at high concentrations cancer cells and also could lead to side effects.
(ca. 100–135 mmol Lꢁ1) and can be taken up from the blood via
In vitro cytotoxicity assays and cellular uptake of DOX-unloaded
MCM-41-COO-P[5]A and DOX-loaded MCM-41-COO-DOX-P[5]A
and free DOX
transporters found in many different organs, such as the
kidneys, intestine, liver, placenta and brain.68–70 Therefore, at
pH 7.4 the citrate anion could compete with the carboxylate
groups anchored on the surface of MCM-41-COOH leading to The cell viability of MCF-7 cells (human breast adenocarcinoma
undesirable release of DOX in the blood and in healthy cells. To cell line) upon treatment with DOX-loaded and DOX-unloaded
check if citrate anions can trigger DOX release from MCM-41- MCM-41-COO-P[5]A and free DOX was assessed. The DOX-unloaded
COO-DOX-P[5]A, we added sodium citrate (19 mmol Lꢁ1) to a nanocarrier was incubated at concentrations of 23.25, 46.50, 116.28
MCM-41-COO-DOX-P[5]A dispersion in PBS buffer. In the first or 232.56 mg mLꢁ1 (Fig. S20, ESI†), which correspond to DOX
two hours of the experiment, no citrate was added and a concentrations in the MCM-41-COO-DOX-P[5]A nanocarrier of 0.5,
minimum amount of DOX was released as previously discussed 1.0, 2.5, and 5.0 mg mLꢁ1, respectively. The cell viability of free DOX
(Fig. 3). After this period, sodium citrate was added and only was evaluated at the same concentrations for comparison.
5% of DOX was released at pH 7.4 (Fig. 5 and Fig. S19, ESI†).
The incubation of MCF-7 cells with DOX-unloaded MCM-41-
In addition, the human body has several metal ions which COO-P[5]A showed some cellular response with a maximum
are essential for organism functioning. Among them, Zn2+ plays decrease of about 50% in cell viability related to the control
vital biological roles,71,72 and can be found in organs, tissues, cells in both 24 and 48 h of incubation time (Fig. 6a and b and
cytoplasm and blood either as a free ion or bound to proteins. Fig. S20, ESI†). However, the impact on MCF-7 viability was
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 6 MTT viability assay of MCF-7 cells treated with DOX-unloaded MCM-41-COO-P[5]A or DOX-loaded MCM-41-COO-DOX-P[5]A or free DOX for
(a) 24 h and (b) 48 h. To reach concentrations of 0.5, 1.0, 2.5 and 5.0 mg mLꢁ1 of DOX in the nanocarrier 23.25, 46.50, 116.28 or 232.56 mg mLꢁ1 of MCM-
41-COO-DOX-P[5]A was used.
neither time- nor concentration-dependent, suggesting that the Most likely, the cytotoxicity improvement caused by the DOX-
phenomenon observed might be due to an initial protective loaded MCM-41-COO-DOX-P[5]A in comparison with free DOX
cellular response rather than a cytotoxic effect of the DOX- might be due to higher cellular uptake through endocytosis
unloaded MCM-41-COO-P[5]A system. In contrast, incubation compared to passive diffusion of free DOX.75,76
of MCF-7 cells with increasing concentrations of DOX-loaded
The MCF-7 cell uptake of MCM-41-COO-DOX-P[5]A and free
MCM-41-COO-DOX-P[5]A revealed a progressive decrease of cell DOX after incubation for 4 and 24 h was also investigated
viability from 40% (0.5 mg mLꢁ1 of DOX) to 25% (5.0 mg mLꢁ1 (Fig. 7). The internalization and location of DOX are indicated
of DOX) in 24 h and from 18% (0.5 mg mLꢁ1 of DOX) to 10% by its red fluorescence. The MCF-7 cell images incubated with
(5.0 mg mLꢁ1 of DOX) in 48 h of treatment (Fig. 6a and b). free DOX at concentrations of 1.0 and 5.0 mg mLꢁ1 show that
Therefore, the MCF-7 treatment with the DOX-loaded MCM-41- DOX is mainly localized in the nucleus of the cells after 4
COO-DOX-P[5]A induces concentration and time-dependent cyto- and 24 h of incubation. Upon treatment of MCF-7 cells with
toxicity. The lowest cell viability was obtained using 2.5 mg mLꢁ1 of DOX-loaded MCM-41-COO-DOX-P[5]A nanocarrier concentra-
DOX loaded in the MCM-41-COO-DOX-P[5]A nanocarrier. Impor- tions of 46.50 and 232.56 mg mLꢁ1, which correspond to 1.0
tantly, the cell viability of the DOX-loaded MCM-41-COO-DOX-P[5]A and 5.0 mg mLꢁ1 of DOX, respectively, for 4 h, one can see that
was lower than that of free DOX at all concentrations investigated. DOX is distributed between the cell membrane and the nucleus
Fig. 7 Confocal fluorescence microscopy images of MCF-7 cells at 37 1C treated with MCM-41-COO-DOX-P[5]A (containing 1.0 and 5.0 mg mLꢁ1 of
DOX) and free DOX (1.0 and 5.0 mg mLꢁ1) for 4 h and 24 h. Red fluorescence: DOX. Blue fluorescence: DAPI (nuclear staining).
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
(labelled with blue fluorescence). After 24 h of treatment, the hydrochloric acid (HCl) (37% PA), sulfuric acid (H2SO4) (98% PA)
drug is mainly detected inside the nucleus of the cells, suggesting and sodium hydroxide (NaOH) (Z99%) were purchased from Vetec.
that MCM-41-COO-DOX-P[5]A is internalized by the MCF-7 cells.
Characterization
1H and 13C NMR spectra of the quaternary ammonium pillar[5]arene
salt were recorded on a Varian VNMRS at 500 MHz and 75 MHz,
Conclusions
respectively, using residual solvent as an internal standard. The
In conclusion, we have designed and constructed a novel pH-
sample was prepared using D2O purchased from Cambridge Isotope
responsive nanocarrier loaded with DOX by integrating negatively
Laboratories.
charged mesoporous silica nanoparticles and a water-soluble catio-
A PerkinElmer CHN 240 C analyser at the Analytical Center
nic pillar[5]arene (P[5]A) nanogate through electrostatic interactions
˜
of the Institute of Chemistry, University of Sao Paulo, Brazil,
between the silanolate groups of MCM-41 (MCM-41-DOX-P[5]A) or
carboxylate groups anchored on the surface of MCM-41 (MCM-41-
COO-DOX-P[5]A) and P[5]A. SEM and TEM images revealed
spherical particles with a narrow size distribution and parallel
nanochannels of MCM-41 and MCM-41-COOH synthesized in
this work. The interactions between the quaternary ammonium
moieties of P[5]A and the carboxylate or silanolate groups on the
mesoporous silica nanoparticles were demonstrated by z-potential
measurements at different pH values. P[5]A was efficient as a
nanogate to block the entrapped drug in the nanochannels of
the MSNPs under physiological conditions. However, for MCM-41-
DOX-P[5]A, P[5]A is trapped inside the nanochannels even under
very acidic conditions, and the nanogate is not completely released.
At pH 2.0, the amount of DOX released is higher than at pH 5.5 for
both nanocarriers. In addition, MCM-41-COO-DOX-P[5]A showed
potential smart on–off release behaviour because of the reversible
nature of the electrostatic interactions. Ionic species such as
citrate3ꢁ and Zn2+ did not show a significant effect on the nanogate
operation at the investigated concentrations. In vitro experiments
showed that the cell viability of DOX-unloaded MCM-41-COO-P[5]A
was neither time- nor concentration-dependent. DOX-loaded MCM-
41-COO-DOX-P[5]A presented a higher cytotoxic effect than free
DOX at the same concentration. Confocal fluorescence microscopy
revealed that the nanocarrier was capable of releasing DOX in the
cell nucleus. Therefore, this novel nanocarrier appears to be a
potential candidate to be envisioned as an alternative system for
cancer therapy; however, additional studies could be very useful to
further evaluate its anticancer potential.
was used to determine the carbon, hydrogen and nitrogen
contents of the MCM-41-CN and MCM-41-COOH samples.
Fourier transform infrared (FTIR) spectra were acquired
using a Thermo Nicolet iS50 FTIR instrument.
Raman spectra were recorded on a Bruker MultiRAM FT-Raman
spectrometer (Bruker, Germany) equipped with a neodymium-
doped yttrium aluminium garnet (Nd:YAG) laser excitation source
operating at lNd:YAG = 1064 nm and a germanium detector. The
samples were placed on the hemispheric bore of an aluminium
sample holder, and the spectra were acquired over the range of
4000–500 cmꢁ1 at room temperature with 128 scans at a laser
power of 300 mW. The spectral resolution was 2 cmꢁ1
Powder X-ray diffraction (PXRD) data of the samples were
collected on a Rigaku Miniflex II X-ray diffractometer with
CuKa (l = 1.5418 Å) within 1.5 and 71 of 2y.
.
Textural analysis was carried out on a Micromeritics ASAP
2020 V304-4 serial 1200 apparatus by determining the N2
adsorption/desorption isotherms at 77 K. Before the analysis,
the samples were heated up to 100 1C at a rate of 1 1C minꢁ1
under a vacuum. The Brunauer–Emmett–Teller (BET) and
Barrett–Joyner–Halenda (BJH) methods were used to assess
the specific surface area and pore size distribution, respectively.
The dynamic light scattering (DLS) size and the zeta
potential (z-potential) were measured using a ZetaPlus Zeta
Potential Analyzer by Brookhaven Instruments Corporation.
The morphological characterization of MCM-41 and MCM-
41-COOH was performed using Scanning Electron Microscopy
(SEM) and High-Resolution Transmission Electron Microscopy
(HRTEM). HRTEM images were acquired on a JEOL 2100F
instrument operated at an accelerating voltage of 200 kV and
a current of 130 mA and equipped with a CCD camera (GATAN
Orius, 11 Mpixel). SEM images were acquired on a field emis-
Experimental
Chemicals
Hexadecyltrimethylammonium bromide (CTAB) (99%), tetra- sion JEOL 7100FT instrument operated at 8 kV with a work
ethyl orthosilicate (TEOS) (99%), 3-cyanopropyltriethoxysilane distance of 9.9 mm and spot size of 8 mm. The samples were
(CyPTES) (Z98%), triphenylphosphine (99%), tetrabromomethane prepared by dropping a sonicated (30 min) ethanolic dispersion
(PA), hydroquinone bis(2-hydroxyethyl)ether (99%), trimethylamine (0.5 mg mLꢁ1) of MCM-41 or MCM-41-COOH onto a copper grid
(35%), paraformaldehyde (PA), boron trifluoride diethyl etherate with a carbon film coating (Ted Pella) and Si substrates for
(PA), doxorubicin chloride (98%), sodium bicarbonate (NaHCO3) HRTEM and SEM analyses, respectively. Then, the grid and Si
(99.7%), sodium sulfate (Na2SO4) (99%), potassium chloride (KCl) substrate were dried at 50 1C for 24 h before analysis. The particle
(99%), sodium chloride (NaCl) (99%), ammonium chloride (NH4Cl) distribution from SEM images was obtained by measuring the
(98%), sodium phosphate dibasic (Na2HPO4) (98%), potassium diameter of about 200 particles using the ImageJ software.
phosphate monobasic (NaH2PO4) (98%), zinc chloride (ZnCl2)
UV-vis absorption spectra were acquired using a Cary 60
(98%), sodium citrate (99%) and 3-(4,5-dimethylthiazol-2-yl)- UV-vis spectrophotometer (Varian). A thermoelectric (Peltier
2,5-diphenyltetrazolium bromide (MTT) were purchased from system) cuvette holder (TLC 40) coupled with a temperature
Sigma-Aldrich and used as received. All necessary solvents and controller (Quantum Northwest TC 125) was used to keep the
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Journal of Materials Chemistry B
Paper
temperature of MCM-41-COO-DOX-P[5]A and MCM-41-DOX- remained under reflux for 2 h. The solid was collected by
P[5] dispersions at 37 1C during DOX release experiments.
filtration, and washed with hot DI water (2ꢃ) and EtOH (1ꢃ).
The organic template extraction procedure was repeated one
more time to remove CTAB completely from the MCM-41 pores,
and then the product was dried yielding a white solid (180 g).
Synthesis of the quaternary ammonium pillar[5]arene salt
(P[5]A)
The synthesis of the quaternary ammonium pillar[5]arene salt
was carried out as described in the literature with minor
modifications.39 Under stirring and a N2 atmosphere, tetrabromo-
Preparation of carboxyl-functionalized MCM-41
(MCM-41-COOH)
methane (23.81 mmol) was slowly added to a mixture containing The mesoporous MCM-41 modification with carboxyl groups
hydroquinone bis(2-hydroxyethyl)ether (10 mmol) and triphenyl- was carried out as described using a methodology employed by
phosphine (21.81 mmol) in 50 mL of dry acetonitrile at 0 1C. After our group to modify silica nanoparticles with various functional
the reaction mixture reached room temperature, it was stirred for groups.17,18,43–49 However, in the procedure used in this work,
an additional 4 h. Then, 40 mL of cold water was added; the the organic template was removed after functionalization with
product precipitated as a white solid and was collected by vacuum 3-cyanopropyl groups. For the functionalization, 2.5 mL of
filtration, washed 3 times with methanol/water and then recrystal- CyPTES was added dropwise to a suspension of MCM-41-
lized from methanol. The white crystals were dried under a CTAB (544 mg) in anhydrous PhMe (200 mL) under reflux in
vacuum, yielding compound 1 (90%) as a white solid. The com- a N2 atmosphere, while stirring vigorously. After 2.0 h, a
pound was characterized by 1H and 13C NMR, and the data fraction of 2.0 mL of PhMe containing EtOH was distilled off
obtained are in agreement with the literature.77 To a solution of from the suspension, followed by the addition of 1.5 mL more
compound 1 in dichloromethane (230 mL), paraformaldehyde of CyPTES. The reaction mixture was left to stir under reflux in
(30.87 mmol) and boron trifluoride diethyl etherate (33.86 mmol) a N2 atmosphere for an additional 24 h. The solid was collected
were added under a N2 atmosphere, and the solution was stirred at by filtration, washed with anhydrous PhMe (1ꢃ) and EtOH (1ꢃ)
room temperature for 2 h. Then, the reaction mixture was washed and dried overnight under air. The obtained material was
with water, saturated sodium bicarbonate solution and brine, dispersed in an ethanolic solution of NH4Cl (5.0 g, 300 mL)
and the organic layer was dried over anhydrous sodium sulfate under vigorous stirring and then the dispersion remained
and concentrated under a vacuum. The residue was purified by under reflux for 2 h. After this period, the solid was filtered,
chromatography on silica gel using dichloromethane/hexane washed with hot DI water (2ꢃ) and EtOH (1ꢃ) and dried
(27 : 23) as the eluent, yielding 66% compound 2 as a white solid. overnight under air. For the hydrolysis of the 3-cyanopropyl
The compound was characterized by 1H and 13C NMR, and the data groups, the obtained solid was slowly dispersed in 100 mL of a
obtained are in agreement with the literature.77 To a solution of 30% H2SO4 solution (v/v) under stirring. The suspension was
compound 2 (1.19 mmol) in ethanol (100 mL), trimethylamine left to stir under reflux for 4 h and the resulting solid was then
(48.11 mmol) was added, and the reaction was stirred overnight collected by filtration, washed with DI water (up to pH = 7) and
under reflux. Then, the mixture was filtered under a vacuum, and dried under a vacuum at RT for 8 h to afford MCM-41-COOH
the solid obtained was washed with ethanol. The product was dried (223 mg).
under a vacuum, yielding compound P[5]A (95%). Mp: 101–103 1C.
1H NMR (D2O, RT, 500 MHz) of P[5]A (ppm): 7.03 (s, 10H), 4.53
Potentiometric titration of MCM-41 and MCM-41-COOH
(s, 20H), 4.02 (s, 10H), 3.88 (s, 20H), 3.31 (s, 90H). 13C NMR (D2O, First, 10.0 mg of MCM-41 or 10 mg of MCM-41-COOH was
RT, 75 MHz) (ppm): 149.43, 129.97, 116.54, 64.93, 63.50, 54.08 dispersed in 10 mL of degassed deionized water by sonication
and 29.54.
for 30 min, and then the dispersion was titrated with
19.86 mmol Lꢁ1 NaOH. The pH of the dispersion was registered
using a pH meter (pH 300 M). The pKa constants of MCM-41
Synthesis of MCM-41-CTAB
The synthesis of MCM-41 was carried out following the procedure and MCM-41-COOH were obtained by the Gran titration
described by Zink et al. with minor modifications.40–42 CTAB (1.0 g) method.59,60
was added to a solution of NaOH in deionized water (DI)
(14.5 mmol Lꢁ1, 967 mL) under vigorous stirring. Subsequently,
MCM-41 and MCM-41-COOH DOX-loading and closing with
P[5]A
the solution was heated at 80 1C for 30 min and after this period
TEOS (10 mL) was added dropwise for approximately 3 min. Then, 64 mg of MCM-41 or MCM-41-COOH was dispersed in 30 mL of
the reaction mixture remained at 80 1C for 2 h. The reaction phosphate buffered saline solution (PBS, pH = 7.4) containing
mixture was cooled to room temperature (RT), filtered, washed 32.2 mg Lꢁ1 of DOX. The dispersions were light protected and
with DI water (2ꢃ) and EtOH (1ꢃ) and dried overnight under air, stirred for 72 h at RT. Then, 32 mg of P[5]A was added to each
yielding a white solid named MCM-41-CTAB (3.0 g).
dispersion, and the mixtures were stirred for 24 h at RT. The
excess of DOX and P[5]A were removed from the resulting
materials through successive centrifugation and washing steps.
Extraction of the organic template from MCM-41-CTAB
To extract the organic template from the pores, MCM-41-CTAB The solids were collected and dried under a vacuum at RT for
(300 mg) was dispersed in an ethanolic solution of NH4Cl (5.0 g, 8 h, resulting in MCM-41-DOX-P[5]A and MCM-41-COO-DOX-
300 mL) under vigorous stirring and then the dispersion P[5]A. The amount of DOX loaded into the nanochannels of
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
MCM-41 or MCM-41-COOH capped by P[5]A was determined by 5.0 mg mLꢁ1 of either free DOX or MCM-41-COO-DOX-P[5]A
a UV-vis standard curve that was created by measuring a series (suspended in PBS, pH 7.4). After treatment for 4 or 24 h, the
of concentrations of DOX solutions in PBS at l = 483 nm. The cells were washed with PBS (pH 7.4) and fixed with parafor-
DOX-loading efficiency was calculated using eqn (1), where M is maldehyde (4%), and the nuclei were stained with DAPI
the initial mass of DOX (1.94 mg) in the solution and m is the (40,6-diamidino-2-phenylindole) (Vectashield). Subsequently,
loaded DOX mass per 100 mg of MCM-41-COOH or MCM-41. the samples were assessed using confocal laser-scanning micro-
The DOX-loading efficiency for MCM-41 and MCM-41-COOH scopy (CLSM, Olympus Fluiview FV 10i, Tokyo, Japan). Analyses
was 52.4% and 57.6%, respectively.
were performed in three independent experiments (DOX: lex
559; lem = 570–670 nm and DAPI: lex = 359; lem = 461 nm).
=
DOX-loading efficiency (%) = m/M ꢃ 100.
(1)
In vitro cytotoxicity assay
MCF-7 cells (9 ꢃ 103 per well) were seeded in a 96-well plate and
cultured in 200 mL of DMEM (supplemented with 10% FBS and
1% penicillin/streptomycin/glutamine) for 24 h at 37 1C under a
humidified 5% CO2 atmosphere. Next, the culture medium was
discarded, and the cells were washed with PBS (pH 7.4). Then,
fresh medium was added with one of the different treatments
(free DOX or MCM-41-COO-DOX-P[5]A) at DOX concentrations
of 0.5, 1.0, 2.5 or 5.0 mg mLꢁ1 and incubated for either 24 h or
48 h. Subsequently, the cells were washed with PBS (pH 7.4)
and incubated with 100 mL of a 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) solution (0.5 mg mLꢁ1 in
DMEM) for 2 h. Then, the MTT solution was removed, and the
cells were lysed with 100 mL of DMSO. The absorption was
measured at l = 570 nm after automatic shaking for 40 s in a
microplate reader (Molecular Devices, Spectra max 190, California,
United States). To determine the biocompatibility of MCM-41-COO-
P[5]A, MCF-7 cells were incubated with increasing amounts of
this material (23.25, 46.50, 116.28 or 232.56 mg mLꢁ1) for 24 or
48 h, and the cell viability was evaluated by incubation with MTT,
as described before. All determinations were performed in three
independent experiments.
DOX controlled release studies
The controlled release of DOX and P[5]A from MCM-41-DOX-
P[5]A and MCM-41-COO-DOX-P[5]A was investigated at different
pH values (2.0, 5.5 and 7.4) or with adding ZnCl2 (1.2 and
50 mmol Lꢁ1) or sodium citrate (19 mmol Lꢁ1) at 37 1C by
UV-vis spectroscopy. All DOX and P[5]A release experiments were
performed by transferring 1 mg of MCM-41-DOX-P[5]A or MCM-
41-COO-DOX-P[5]A to a quartz cuvette containing a small magnetic
stir bar and adding 2 mL of a PBS solution (pH = 7.4). The cuvettes
were placed into a thermoelectrically controlled cuvette holder
adjusted to 37 1C and gently stirred. After the decantation of the
solid, UV-vis spectra were recorded at different time intervals up to
30 h. The DOX and P[5]A release was trigged by changing the pH of
the PBS suspension (pH = 7.4) to 5.5 and 2.0 by adding calculated
volumes of HCl solution (3.0 mol Lꢁ1).
Chemical switching experiments (ON–OFF)
The changes in the absorbance of DOX were observed at
483 nm for MCM-41-COO-DOX-P[5]A (1 mg) dispersed in a
PBS solution (2 mL) upon the successive addition of calculated
volumes of acid (3.0 mol Lꢁ1 HCl) to reach pH 5.5 or 2.0 and
base (NaOH 3.0 mol Lꢁ1) to return to pH 7.4. The dilutions were
considered to calculate the final concentrations of DOX.
Conflicts of interest
There are no conflicts to declare.
Cell lines and treatments
Human breast adenocarcinoma cells (MCF-7) were obtained from
the Laboratory of Cellular and Molecular Hemato-Oncology of the
Brazilian National Cancer Institute and cultured in Dulbecco’s
Acknowledgements
modified Eagle’s medium (DMEM) (Sigma) containing 10% fetal The authors gratefully acknowledge the National Council for
bovine serum (FBS) and 1% penicillin/streptomycin/glutamine Scientific and Technological Development for the research
(Invitrogen) at 37 1C under 5% CO2. Doxorubicin hydrochloride grant (CNPq grant number 434141/2018-6 and 313799/2017-2)
was added to the culture medium of exponentially growing cells at and fellowships of C. M. R. and L. F. R. P.; the Brazilian Federal
increasing concentrations and time periods. 3-(4,5-Dimethylthiazol- Agency for the Support and Evaluation of Graduate Education
2-yl)-2,5-diphenyltetrazolium bromide (MTT) was also added to the (CAPES) for the fellowships of E. C. S. S., T. C. S. and T. S. F. and
´
cell culture medium.
Pro-equipamentos, and the Rio de Janeiro Research Founda-
tion for the fellowship of V. G. C. M and research projects
(Faperj grant number E-26/010101133/2018 and E-26/202629/
In vitro cellular uptake
For the cellular uptake of DOX-loaded MCM-41-COO-DOX-P[5]A 2019). We are grateful to the Material Characterization (http://
and free doxorubicin (DOX) MCF-7 cells (9 ꢃ 103 cells per well) www.uff.br/lamate/), Molecular Spectroscopy (http://www.uff.
were grown on coverslips in a 24-well plate with 500 mL of br/lame/), NMR (http://www.laremn.uff.br), and Multiuser
DMEM (supplemented with 10% FBS and 1% penicillin/strep- Laboratories from Universidade Federal Fluminense (UFF).
tomycin/glutamine) for 24 h at 37 1C under 5% CO2. Then, the The authors also would like to thank the Multiuser Laboratory
cells were rapidly washed with PBS (pH 7.4), and the culture of Nanoscience and Nanotechnology from Centro Brasileiro de
´
medium was replaced by fresh medium containing 1.0 or Pesquisas Fısicas (LABNANO-CBPF).
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Journal of Materials Chemistry B
Paper
27 T. Ogoshi, M. Hashizume, T. Yamagishi and Y. Nakamoto,
Chem. Commun., 2010, 46, 3708–3710.
28 N. Song, T. Kakuta, T. Yamagishi, Y.-W. Yang and T. Ogoshi,
Chem, 2018, 4, 2029–2053.
Notes and references
1 R. Sun, W. Wang, Y. Wen and X. Zhang, Nanomaterials,
2015, 5, 2019–2053.
2 C. R. Thomas, D. P. Ferris, J.-H. Lee, E. Choi, M. H. Cho,
E. S. Kim, J. F. Stoddart, J.-S. Shin, J. Cheon and J. I. Zink,
J. Am. Chem. Soc., 2010, 132, 10623–10625.
29 X.-Y. Lou, Y.-P. Li and Y.-W. Yang, Biotechnol. J., 2018,
14, 1800354.
30 X. Wu, Y. Zhang, Y. Lu, S. Pang, K. Yang, Z. Tian, Y. Pei,
Y. Qu, F. Wang and Z. Pei, J. Mater. Chem. B, 2017, 5,
3483–3487.
31 Y.-L. Sun, Y.-W. Yang, D.-X. Chen, G. Wang, Y. Zhou, C.-Y.
Wang and J. F. Stoddart, Small, 2013, 9, 3224–3229.
32 X. Wang, L.-L. Tan, X. Li, N. Song, Z. Li, J.-N. Hu, Y.-M.
Cheng, Y. Wang and Y.-W. Yang, Chem. Commun., 2016, 52,
13775–13778.
33 H. Li, R. Wei, G.-H. Yan, J. Sun, C. Li, H. Wang, L. Shi,
J. A. Capobianco and L. Sun, ACS Appl. Mater. Interfaces,
2018, 10, 4910–4920.
´
3 J. S. F. Silva, J. Y. R. Silva, G. F. de Sa, S. S. Arau´jo, M. A. G.
Filho, C. M. Ronconi, T. C. Santos and S. A. Ju´nior, ACS
Omega, 2018, 3, 12147–12157.
4 X. Huang and X. Z. Du, ACS Appl. Mater. Interfaces, 2014, 6,
20430–20436.
´
´
5 E. Aznar, M. Oroval, L. Pascual, J. R. Murguıa, R. Martınez-
´˜
´
Manez and F. Sancenon, Chem. Rev., 2016, 116, 561–718.
6 L.-B. Meng, W. Zhang, D. Li, Y. Li, X.-Y. Hu, L. Wang and
G. Li, Chem. Commun., 2015, 51, 14381–14384.
7 X. Wu, Y. Li, C. Lin, X.-Y. Hu and L. Wang, Chem. Commun.,
2015, 51, 6832–6835.
34 L.-L. Tan, H. Li, Y.-C. Qiu, D.-X. Chen, X. Wang, R.-Y. Pan,
Y. Wang, S. X.-A. Zhang, B. Wang and Y.-W. Yang, Chem.
Sci., 2015, 6, 1640–1644.
35 L.-L. Tan, H. Li, Y. Zhou, Y. Zhang, X. Feng, B. Wang and
Y.-W. Yang, Small, 2015, 11, 3807–3813.
8 N. Song and Y.-W. Yang, Chem. Soc. Rev., 2015, 44, 3474–3504.
9 Z. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink,
Chem. Soc. Rev., 2012, 41, 2590–2605.
10 Y. Xu, Y. Zhu and S. Kaskel, RSC Adv., 2015, 5, 99875–99883.
11 B. Ru¨hle, S. Datz, C. Argyo, T. Bein and J. I. Zink, Chem.
Commun., 2016, 52, 1843–1846.
36 X. Huang, S. Wu, X. Ke, X. Li and X. Du, ACS Appl. Mater.
Interfaces, 2017, 9, 19638–19645.
12 D. Tarn, D. P. Ferris, J. C. Barnes, M. W. Ambrogio,
J. F. Stoddart and J. I. Zink, Nanoscale, 2014, 6, 3335–3343.
13 C. D. Ding, Y. Liu, T. Wang and J. J. Fu, J. Mater. Chem. B,
2016, 4, 2819–2827.
14 Y. Yao, Y. Wang, R. Zhao, L. Shao, R. Tang and F. Huang,
J. Mater. Chem. B, 2016, 4, 2691–2696.
37 Q.-L. Li, Y. Sun, L. Ren, X. Wang, C. Wang, L. Li, Y.-W. Yang,
X. Yu and J. Yu, ACS Appl. Mater. Interfaces, 2018, 10,
29314–29324.
38 M.-X. Wu, J. Gao, F. Wang, J. Yang, N. Song, X. Jin, P. Mi,
J. Tian, J. Luo, F. Liang and Y.-W. Yang, Small, 2018,
14, 1704440.
15 P.-J. Chen, S.-H. Hu, C.-S. Hsiao, Y.-Y. Chen, D.-M. Liu and
S.-Y. Chen, J. Mater. Chem., 2011, 21, 2535–2543.
16 Y.-L. Sun, Y. Zhou, Q.-L. Li and Y.-W. Yang, Chem. Commun.,
2013, 49, 9033–9035.
17 G. Q. Silveira, R. S. Da Silva, L. P. Franco, M. D. Vargas and
C. M. Ronconi, Microporous Mesoporous Mater., 2015, 206,
226–233.
39 Y. Ma, X. Ji, F. Xiang, X. Chi, C. Han, J. He, Z. Abliz, W. Chen
and F. Huang, Chem. Commun., 2011, 47, 12340–12342.
40 W. Chen, C.-A. Cheng, B.-Y. Lee, D. L. Clemens, W.-Y.
Huang, M. A. Horwitz and J. I. Zink, ACS Appl. Mater.
Interfaces, 2018, 10, 31870–31881.
41 B. Ruehle, D. L. Clemens, B.-Y. Lee, M. A. Horwitz and
J. I. Zink, J. Am. Chem. Soc., 2017, 139, 6663–6668.
42 W.-Y. Huang and J. I. Zink, J. Phys. Chem. C, 2016, 120,
23780–23787.
18 G. Q. Silveira, M. D. Vargas and C. M. Ronconi, J. Mater.
Chem., 2011, 21, 6034–6039.
19 T. D. Nguyen, K. C.-F. Leung, M. Liong, C. D. Pentecost,
J. F. Stoddart and J. I. Zink, Org. Lett., 2006, 8, 3363–3366.
20 Y. Zhou, L.-L. Tan, Q.-L. Li, X.-L. Qiu, A.-D. Qi, Y. Tao and
Y.-W. Yang, Chem. – Eur. J., 2014, 20, 2998–3004.
21 Z. Tong, J. Zhou, R. Huang, J. Zhou, R. Zhang, W. Zhuo and
G. Jiang, J. Polym. Sci., Part A: Polym. Chem., 2017, 55, 2477–2482.
22 T. Ogoshi, S. Kanai, S. Fujinami, T.-A. Yamagishi and
Y. Nakamoto, J. Am. Chem. Soc., 2008, 130, 5022–5023.
23 T. Ogoshi and T. Yamagishi, Chem. Commun., 2014, 50,
4776–4787.
˜
43 E. C. S. Santos, T. C. dos Santos, R. B. Guimaraes, L. Ishida,
R. S. Freitas and C. M. Ronconi, RSC Adv., 2015, 5,
48031–48038.
44 M. R. Mello, D. Phanon, G. Q. Silveira, P. L. Llewellyn and
C. M. Ronconi, Microporous Mesoporous Mater., 2011, 143,
174–179.
45 A. L. de Lima, C. M. Ronconi and C. J. A. Mota, Catal. Sci.
Technol., 2016, 6, 2877–2891.
46 T. C. dos Santos, S. Bourrelly, P. L. Llewellyn, J. W. M.
Carneiro and C. M. Ronconi, Phys. Chem. Chem. Phys., 2015,
17, 11095–11102.
24 P. J. Cragg and K. Sharma, Chem. Soc. Rev., 2012, 41,
597–607.
25 M. Xue, Y. Yang, X. Chi, Z. Zhang and F. Huang, Acc. Chem.
Res., 2012, 45, 1294–1308.
47 A. L. de Lima, J. S. C. Vieira, C. M. Ronconi and C. J. A. Mota,
Mol. Catal., 2018, 458, 240–246.
48 G. Q. Silveira, C. M. Ronconi, M. D. Vargas, R. A. S. San Gil
26 T. S. Fernandes, E. C. S. Santos, V. G. C. Madriaga,
V. Nascimento, F. Garcia and C. M. Ronconi, J. Braz. Chem.
Soc., 2019, 30, 2452–2463.
˜
and A. Magalhaes, J. Braz. Chem. Soc., 2011, 22, 961–967.
49 A. L. de Lima, A. Mbengue, R. A. S. San Gil, C. M. Ronconi
and C. J. A. Mota, Catal. Today, 2014, 226, 210–216.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
50 T. C. Dos Santos and C. M. Ronconi, Rev. Virtual Quim., 64 E. C. S. Santos, A. Watanabe, M. D. Vargas, M. N. Tanaka,
2014, 6, 112–130. F. Garcia and C. M. Ronconi, New J. Chem., 2018, 42, 671–680.
51 B. A. Morrow and A. J. Mcfarlan, J. Phys. Chem., 1992, 96, 65 N. V. Roik and L. A. Belyakova, Interface Focus, 2016, 6, 20160041.
1395–1400.
66 L. C. Costello and R. B. Franklin, J. Hum. Endocrinol., 2016,
1, 005.
52 G. Su, C. Yang and J.-J. Zhu, Langmuir, 2015, 31, 817–823.
53 B. Schrader and D. Bougeard, Infrared and Raman Spectro- 67 P. Icard, L. Poulain and H. Lincet, Biochim. Biophys. Acta,
scopy: Methods and Applications, VCH, Weinheim, 1995. 2012, 1825, 111–116.
54 L. G. Thygesen, M. M. Lokke, E. Micklander and S. B. Engelsen, 68 M. E. Mycielska, A. Patel, N. Rizaner, M. P. Mazurek,
Trends Food Sci. Technol., 2003, 14, 50–57.
55 S. Fiorilli, B. Onida, B. Bonelli and E. Garrone, J. Phys. Chem.
B, 2005, 109, 16725–16729.
56 K. S. W. Sing, Pure Appl. Chem., 1985, 57, 603–619.
57 M. Thommes, K. Kaneko, A. V. Neimark, J. P. Olivier,
F. Rodriguez-Reinoso, J. Rouquerol and K. S. W. Sing, Pure
Appl. Chem., 2015, 87, 1051–1069.
H. Keun, A. Patel, V. Ganapathy and M. B. A. Djamgoz,
BioEssays, 2009, 31, 10–20.
69 A. M. Pajor, Pflugers Arch., 2014, 466, 119–130.
70 S. A. Fraenkl, J. Muser, R. Groell, G. Reinhard, S. Orgul,
J. Flammer and D. Goldblum, J. Ocul. Pharmacol. Ther.,
2011, 27, 577–580.
71 K. Kaur, R. Gupta, S. A. Saraf and S. K. Saraf, Compr. Rev.
Food Sci. Food Saf., 2014, 13, 358–376.
´
´
58 N. La-Salvia, J. J. Lovon-Quintana, A. S. P. Lovon and
G. P. Valença, Mater. Res., 2017, 20, 1461–1469.
59 K. Yu, N. Kumar, A. Aho, J. Roine, I. Heinmaa, D. Y. Murzin
and A. Ivaska, J. Catal., 2016, 335, 117–124.
72 R. A. Bozym, R. B. Thompson, A. K. Stoddard and
C. A. Fierke, ACS Chem. Biol., 2006, 1, 103–111.
73 Metals and Genetics, ed. B. Sarkar, Springer US, Boston, MA,
1999.
60 N. D. Shcherban, S. M. Filonenko, R. Y. Barakov, S. A.
Sergiienko, K. Yu, I. Heinmaa, A. Ivaska and D. Y. Murzin, 74 W. Maret, Metallomics, 2015, 7, 202–211.
Appl. Catal., A, 2017, 543, 34–42.
75 A. Hervault and N. T. K. Thanh, Nanoscale, 2014, 6,
61 W. Zhao, J. Chu, F. Xie, Q. Duan, L. He and S. Zhang,
J. Chromatogr. A, 2017, 1485, 44–51.
62 D.-X. Chen, Y.-L. Sun, Y. Zhang, J.-Y. Cui, F.-Z. Shen and
Y.-W. Yang, RSC Adv., 2013, 3, 5765–5768.
63 T. Zhou, H. Yu, M. Liu and Y.-W. Yang, Chin. J. Chem., 2015,
33, 125–130.
11553–11573.
76 M. I. Majeed, Q. Lu, W. Yan, Z. Li, I. Hussain, W. Tremel, M. N.
Tahir and B. Tan, J. Mater. Chem. B, 2013, 1, 2874–2884.
77 E. H. Wanderlind, D. G. Liz, A. P. Gerola, R. F. Affeldt,
V. Nascimento, L. C. Bretanha, R. Montecinos, L. Garcia-Rio,
H. D. Fiedler and F. Nome, ACS Catal., 2018, 8, 3343–3347.
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2019